Metagenomic analysis of endemic viruses in oral secretions from Chinese pigs

Abstract Background Pigs are unique reservoirs for virus ecology. Despite the increased use of improved biosecurity measures, pig viruses readily circulate in Chinese swine farms. Objectives The main objective of this study was to examine archived swine oral secretion samples with a panel of pan‐species viral assays such that we might better describe the viral ecology of swine endemic viruses in Chinese farms. Methodology Two hundred (n = 200) swine oral secretion samples, collected during 2015 and 2016 from healthy pigs on six swine farms in two provinces in China, were screened with molecular pan‐species assays for coronaviruses (CoVs), adenoviruses (AdVs), enteroviruses (EVs), and paramyxoviruses (PMV). Samples were also screened for porcine circovirus (PCV) 3, porcine reproductive and respiratory syndrome virus (PRRSV) and influenza A virus (IAV). Results Among 200 swine oral secretion samples, 152 (76.0%) were found to have at least one viral detection. Thirty‐four samples (17%) were positive for more than one virus, including 24 (70.5%) with dual detection and 10 (29.5%) with triple detection. Seventy‐eight (39.0%) samples were positive for porcine AdVs, 22 (11.0%) were positive for porcine CoVs, 21 (10.5%) were positive for IAVs, 13 (6.5%) were positive for PCV, 7 (3.5%) were positive for PMV, six (3.0%) were positive for PRRSV and five (2.5%) were positive for porcine EV. Conclusion Our findings underscore the high prevalence of numerous viruses among production pigs in China and highlight the need for routine, periodic surveillance for novel virus emergence with the goal of protecting pigs.


INTRODUCTION
Pigs are considered important reservoirs for multiple pathogens and provide unique environments for virus ecology. In the past decade, the world has experienced the emergence and spread of a number of novel pig viruses that occasionally spill over to humans. The rapid expansion of the commercial swine industry has contributed to the emergence and rapid spread of swine viruses that could be a major threat to the pig industry worldwide, including China. A high prevalence of swine viruses is found in large herds, and new variants are routinely being discovered (Hause & Scheidt 2016;Ramesh et al., 2021). Recently, a review article (VanderWaal & Deen, 2018) summarised publications during the last 50 years and identified pigs as hosts harboring many viruses. Mounting evidence suggests that coinfections are more prevalent in modern swine farms than single pathogen infections Gray & Baker, 2011;Guo et al., 2020;Ma et al., 2015;Ma et al., 2018;Saade et al., 2020;Sun et al., 2015). Although several studies have explored swine virus ecology, such studies have seldom examined virus ecology in Chinese swine farms (VanderWaal & Deen, 2018), and fewer still have examined viral coinfections among Chinese pigs.
Among swine pathogens, a variety of infectious agents are shed in oral fluid, including many of the most economically important. Oral fluid sampling is a non-invasive and simple method to study swine pathogens at the herd level (Prickett et al., 2008). In this study, we examined archived swine oral secretion samples collected for influenza A virus (IAV) surveillance  with a panel of panspecies viral assays such that we might better describe the viral ecology of swine endemic viruses in Chinese farms.

Study design
This study was approved by the institutional review boards of Duke University and the Beijing Institute of Microbiology and Epidemiology.
During March 2015, as part of an ongoing 5-year prospective epidemiological study to assess IAV ecology, six Chinese swine farms (three each in Jiangsu and Shandong Provinces) were visited on a monthly basis to collect samples from pigs at different stages of production (growers, finishers, sows and boars). The enrolled farms varied in size (0.6-4 km 2 ), the average number of pigs on-site per day  and the number of swine houses (3-27) Ma et al., 2018). Briefly, data captured for each specimen included the location, age and gender of the pigs in each pen (Table 1). Pig oral secretion (POS) samples were collected using a hanging rope method where three-strand braided unbleached 100% cotton ropes, with 5/8″ diameter, were pre-soaked with a 5% sterile glucose solution and placed in swine pens Ma et al., 2018). Ropes were attached to a rod or pole and placed 40 cm above the ground for 20 to 30 mins during which time the pigs would chew on the rope. At the conclusion of the sampling, oral fluids were manually and aseptically expressed from the rope into a sterile sampling bag (Cat. No. EPR-4590, Labplas, Inc.). Samples were transported under cold chain methods to Chinese public health laboratories where processing and initial IAV molecular assays were conducted.  Xiu et al., 2020) and porcine reproductive and respiratory syndrome virus (PRRSV; Xie et al., 2013). In addition, the viral RNA of each sample was screened for IAV by a qRT-PCR assay targeting the influenza matrix genome segment using a one-step RT-PCR kit (Cat.

Sample selection and laboratory analyses
No. 56046, TaKaRa) on an Applied Biosystems 7500 real-time PCR platform (Life Technologies) as previously described .
All PCR runs had a negative template control (nuclease-free water) and a corresponding synthetic positive control sample included.

Sequencing and phylogenetic analyses
Partial genome sequencing of positive samples was performed by a commercial sequencing company (Genewiz). Assembly and analysis of sequence data were conducted using BioEdit Software version 5.0.9. This program was also used to edit the sequencing electropherograms and to exclude nucleotide ambiguity. Multiple sequence alignments were performed using ClustalW (Thompson et al., 1994).
Sequences were submitted to national center for biotechnology infor- were employed. Sequences were aligned using the neighbour-joining method in MEGA X (Kumar et al., 2018). A bootstrap analysis was performed to assess the confidence limits of the branching with 1000 replicates.

Overall
in different regions of China (Liu et al., 2011). PRRSV and PCV3 coinfection in China has also been previously detected (Chen et al., 2017). Furthermore, CSFV, PRRSV and PCV2 coinfection was also observed in previous studies, with prevalence rates ranging from 2.5% to 3.6% Xu et al., 2012). A more recent study demonstrated that 12.9%, 36.0% and 1.8% of PRRSV-positive pigs were coinfected with PCV2, PRRSV and CSF, respectively .
We also performed phylogenetic analysis of sequence data to understand the diversity of each virus. Alignment of the sequences among the EV strains revealed EV-G (86%-96%). EV-G is prevalent and widespread in the general pig population in middle and eastern China, and infections tend to occur early, usually within the first week after birth (Mi et al., 2021).
None of the PMV-positive samples was successfully sequenced.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest regarding the publication of this article.  -14-08) and the Academy of Military Medical Sciences (AMMS-20-14-009).

DATA AVAILABILITY STATEMENT
None.